Kinin‐B1 and B2 receptor activity in proliferation and neural phenotype determination of mouse embryonic stem cells

Abstract

The kinins bradykinin and des-arg 9 -bradykinin cleaved from kininogen precursors by kallikreins exert their biological actions by stimulating kinin-B2 and B1 receptors, respectively. In vitro models of neural differentiation such as P19 embryonal carcinoma cells and neural progenitor cells have suggested the involvement of B2 receptors in neural differentiation and phenotype determination; however, the involvement of B1 receptors in these processes has not been established. Here, we show that B1 and B2 receptors are differentially expressed in mouse embryonic E14Tg2A stem cells undergoing neural differentiation. Proliferation and differentiation assays, performed in the presence of receptor subtype-selective agonists and antagonists, revealed that B1 receptor activity is required for the proliferation of embryonic and differentiating cells as well as for neuronal maturation at later stages of differentiation, while the B2 receptor acts on neural phenotype choice, promoting neurogenesis over gliogenesis. Besides the elucidation of bradykinin functions in an in vitro model reflecting early embryogenesis and neurogenesis, this study contributes to the understanding of B1 receptor functions in this process. V C 2015 International Society for Advancement of Cytometry Key terms bradykinin; des-arg 9 -bradykinin; kinin receptors; embryonic stem cells; neural differentiation; cholinergic neurons THE kallikrein-kinin system is a multi-protein set involved in several biological processes (1). The kininogens are glycoproteins present in plasma and other biological fluids, which upon cleavage by kallikreins, a subgroup of serine proteases, release bioactive kinins. Kinin actions are mediated by two G-protein coupled receptors, denominated kinin-B1 and B2 receptors, distinguished by their pharmacological properties and biological functions. The B2 receptor has affinity for intact kinins (bradykinin and kallidin), whereas the B1 receptor is activated by the metabolites des-Arg 9 -bradykinin and des-Arg 10 -kallidin originated from the cleavage of the Cterminal arginine of kinins by carboxypeptidases M/N (2,3).In contrast to the B2 receptor that is expressed in a wide variety of cells and tissues, the B1 receptor is poorly expressed under normal conditions, but its expression can be induced in vivo and in vitro by endotoxins, cytokines and growth factors (4). Interestingly, unlike in other tissues, both B1 and B2 receptors, are constitutively expressed in the central nervous system (CNS) (5). These receptors have been found in the cytoplasm of neuronal cell bodies in the hypothalamus, thalamus, and the frontal cortex, while only the B1 receptor was expressed in ependymal cells of the choroid plexus (6,7).

Bradykinin promotes neuron-generating division of neural progenitor cells through ERK activation

“…Previous studies have shown that bradykinin induces an elevation in the concentration of intracellular free Ca 2+ ([Ca 2+ ] i ) by activating B2BkR in four models of neural differentiation (Appell and Barefoot, 1989;Martins et al, 2008Martins et al, , 2005Nascimento et al, 2015;Trujillo et al, 2012). Thus, we aimed to find the intracellular signaling pathways that mediate the bradykinin-induced [Ca 2+ ] i increase.…”

Glioblastoma‐mesenchymal stem cell communication modulates expression patterns of kinin receptors: Possible involvement of bradykinin in information flow

“…Cells were passed through a 40 mm cell strainer, centrifuged, then fixed for 20 min in 4% paraformaldehyde (PFA) at room temperature (RT), washed with PBS supplemented with 2% FBS, and incubated for 30 min at RT with mouse anti-B2R (1:100; BD Biosciences) and rabbit anti-B1R (1:100; Abcam) primary antibodies in PBS plus 0.1% Triton X-100 and 2% FBS. After washing with 900 ml of PBS plus 2% FBS, the secondary anti-mouse antibodies conjugated with Alexa Fluor (AF) 647 and anti-rabbit conjugated antibodies with AF 488 (both at 1:500 dilutions; Life Technologies) were added and incubated for 30 min at RT (18,19). After a final washing step, the cells were re-suspended in 400 ml of PBS and analyzed on a flow cytometer (BD Accuri C6, BD Biosciences).…”

Kinins and microglial responses in bipolar disorder: a neuroinflammation hypothesis

“…More recently, studies have demonstrated the role of BK in neurite outgrowth, proliferation inhibition of stem cells and their differentiation into neurons (Martins et al, 2005;Trujillo et al, 2009;Trujillo et al, 2012;Nascimento et al, 2015;Pillat et al, 2015). At a molecular level, BK and B2BKR are also involved in ERK activation, acting via tyrosine kinases Pyk2 and Src (Ca 2+ -dependent pathway), and via EGF and PKC pathway, respectively (Dikic et al, 1996;Adomeit et al, 1999).…”